Title: Development of Compact CRISPR Editors for Multiplexed Editing of Complex Gene Networks Abstract The ability to perform multiplexed editing of the genome will provide significant opportunity to investigate into the interplay of various elements in the complex gene network. Since its advent, the CRISPR-Cas system has demonstrated unique advantages over previous genomic editing tools such as sequence specificity, programmability, and the ease of implementation. However, the utility of the existing non-nuclease CRISPR editors is still hampered by major challenges including limited ability of multiplexing and inefficient intracellular delivery. In this proposal, we aim to develop a new class of compact, multiplexable CRISPR editors that allow the genomic editing at transcriptional, epigenetic, and post-translational levels. At the center of these CRISPR editors are intracellularly evolved RNA aptamers that are capable of recruiting endogenous effectors independent of additional labeling and tagging, thereby enabling the locus-specific co-localization of the CRISPR editors and the endogenous effectors. CRISPR-based constructs will be developed to enable the intracellular selection of aptamers in both bacterial and mammalian cells for robust and highly specific recognition of various effectors regulating distinct cellular functions. We will further implement this technology into the multiplexable interrogation and manipulation of multiple distinct cellular functions including transcriptional activation, epigenetic remodeling, and DNA nucleobase editing. Compared to the existing CRISPR editors, the new CRISPR editors developed in this proposal do not require the delivery of exogenous effectors, allowing for the encoding gene to be efficiently delivered by a single adeno-associated virus (AAV) vector. These CRIPSR editors will be applied to executing multiplexed editing programs in genome-scale screens to identify novel genetic elements responsible for the emergence of drug resistance in cancer. If successful, the tools developed in this proposal can significantly improve our ability to understand and engineer the gene network in various applications spanning fundamental research and therapeutics.